The present invention relates to tumor cells having tumorigenic potential but lacking invasive/metastatic potential, a method for preparing them and a screening method for metastasis-related genes using them.
Invasion and metastasis occur at the final stage of tumor progression and are the major cause of death of cancer patients. However, it is difficult to treat such effects because the molecular mechanism by which malignant phenotypes of tumor cells such as invasion/metastasis are expressed has been mostly unknown. Although the expression of malignant phenotypes such as invasion/metastasis has long been recognized as a biological phenomenon which is pathologically distinguished from tumor formation in carcinogenic process, there has been only limited information about biochemical basis therefor and target groups of molecules for therapy have not been well identified.
Generally, accumulation of alterations of genetic information of oncogenes and tumor suppressor genes is thought to make cells cancerous and sometimes invasive/metastatic. Namely, a gene that plays a role in mediating an external signal into the nucleus is mutated to form a gene product, which activates the downstream signaling cascade continuously. This causes phenotypes of tumor cells to be expressed. It is also thought that tumor cells become invasive/metastatic during tumor progression as a result of additional genetic alterations to those required for tumorigenicity (Evans C. W. The Metastatic Cell: Behavior and Biochemistry, Chapman and Hall, London, 1991). Therefore, metastasis of tumor cells may be induced by an aberrant intracellular signaling system that is different from the signaling system required for tumor-igenicity. Thus, it is very important for investigations of the mechanism of invasion/metastasis to elucidate a metastasis-specific tumor-related signaling pathway that is responsible for tumor cells becoming invasive/metastatic.
For such investigations, a cell which could separably acquire tumorigenic potential and invasive/metastatic potential by oncogene transfer would be useful for identifying the above pathway and molecules involved therein.
A set of two cells derived from the same parent strain, one of which has tumorigenic potential but lacks invasive/metastatic potential while the other has both potentials, would be useful for investigating the signaling pathway involved in metastasis. Some tumor cell lines satisfying this criterion have already been established. They include rodent-derived cell lines K1735, B16, T-lymphoma L5178Y, Lewis lung carcinoma and rat ascites hepatoma AH7974, as well as their derivatives obtained by in vitro culture and selection of cells recovered from in vivo metastases of these cell lines (Fidler I. J. et al., J. Natl. Cancer Inst. 67:947-956, 1981; Talmadge J. E. et al., J. Natl. Cancer Inst. 69:975-980, 1982; Fidler I. J., Nature 242:148-149, 1973; Fidler I. J., Cancer Res. 35:218-224, 1975; Fidler I. J. et al., Am. J. Pathol. 97:633-648, 1979; Ota et al., Clin. Exp. Metastasis 10:297-308, 1992; Olsson L. et al., Cancer Res. 41:4706-4709, 1981; Brodt P., Cancer Res. 46:2442-2448, 1986; Pal. K. et al., Invasion Metastasis 5:159-169, 1985; Young M. R. et al., Cancer Res. 45:3918-3923, 1985; Kawaguchi T. et al., Clin. Exp. Metastasis 10:225-238, 1992). However, phenotypes of these cell lines are sometimes unstable during experimentations (Poste G. et al., Proc. Natl. Acad. Sci. USA 78:6226-6230, 1981) and many properties of these cell lines that are not related to the acquisition of invasive/metastatic potential may change during in vivo selection.
It would be useful and valuable if a stable set of cell lines as described above could be obtained by oncogene transfer. However, whether or not cells acquire tumorigenic potential and invasive/metastatic potential by oncogene transfer depends on the nature of the cell line used (Muschel R. J. et al., Am. J. Pathol. 121:1-8, 1985). Moreover, oncogene transfer frequently induces invasive/metastatic potential in recipient cells to a greater or lesser extent simultaneously with tumorigenicity (Thorgeirsson U. P., Mol. Cell. Biol. 5:259-262, 1985; Egan S. E. et al., Science 238:202-205, 1987). Up to the present, no report has shown exact establishment of a cell line having tumorigenic potential but lacking invasive/metastatic potential and a cell line having both of these potentials. Thus, it was not easy to separately use characteristics of tumorigenic potential and invasive/metastatic potential of tumor cells for investigations in an experimental system.
It is an object of the present invention to establish a tumor cell line having tumorigenic potential but lacking invasive/metastatic potential, which is suitable for investigating the difference between the signaling pathway for acquiring tumorigenic potential and the signaling pathway for acquiring invasive/metastatic potential. Such a tumor cell line can be used in combination with a cell line derived from the same parent strain but having both of tumorigenic and invasive/metastatic potentials for investigations of characteristics of invasive/metastatic potential. It is another object of the present invention to develop a screening method for tumor metastasis-related genes using the tumor cell line of the present invention.
As a result of careful studies to overcome the above problems, we succeeded in establishing a tumor cell line having tumorigenic potential but lacking invasive/metastatic potential by introducing a specific oncogene into a recipient cell.
Accordingly, the present invention provides a tumor cell having tumorigenic potential but lacking invasive/metastatic potential. A preferred tumor cell of the present invention having such properties is 1-1ras1000 deposited with the National Institute of Bioscience and Human-Technology of the Agency of Industrial Science and Technology under accession number FERM BP-5406.
The deposit was made on Feb. 20, 1996 and the address of the National Institute of Bioscience and Human Technology Agency of Industrial Science and Technology, Ministry of International Trade and Industry, is 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305, Japan.
The present invention also provides a method for preparing said tumor cell having tumorigenic potential but lacking invasive/metastatic potential by introducing an oncogene of the ras family into a BALB/c 3T3 A31 variant cell.
The present invention also provides a screening method for genes having the property of conferring invasive/metastatic potential, which comprises transfecting DNA derived from a tumor tissue obtained from the surface or inside of a mammal or derived from a tumor cell line into a tumor cell having tumorigenic potential but lacking invasive/metastatic potential, isolating cells having acquired invasive/metastatic potential and extracting DNA therefrom.